This invention relates to a method of controlling the population of a first microorganism at a foliar locus by preferentially enhancing the population of a second microorganism at said locus.
Microbiological plant pathogens and insect pests of plants represent a major source of loss of agricultural productivity throughout the world. Research on the biological control of foliar pathogens is limited when compared with studies concerning this form of control for soilborne pathogens. However, as restrictions on the use of chemical pesticides increase, biological control of foliar pathogens is becoming more important.
The ecosystems which exist on above-ground plant surfaces are complex, and the microbial interactions which occur on it are poorly understood. Plant pathogens and their interactions with the host plant have been influenced under limited circumstances by the population of microbial epiphytes which exist in the foliar phylloplane. The introduction of organisms that exhibit antagonism towards foliar pathogens in this environment has been successful in controlling certain pathogens by several mechanisms, which include direct parasitism of the pathogen by the epiphyte, competition for nutrients and space, the production of antibiotics and/or lytic enzymes by the antagonist, and stimulation of a host resistance response.
Recently, there have been several reported successes using introduced antagonists for the control of foliar diseases in the field. Spurr (1981) used Pseudomonas cepacia to inhibit the germination of Alternaria alternata and thereby control Alternaria leaf spot of tobacco in field tests by repeated applications over a period of three years. Limited but significant control of Cercospora leaf spot on peanut was achieved in field tests using aqueous suspensions of P. cepacia applied to foliage at two week intervals. In later experiments by Bailey and Spurr (1984), P. cepacia and Bacillus thuringiensis (Bt) were formulated as wettable powders and sprayed onto peanut foliage using xanthan gum, a water-soluble polysaccharide, as a sticking agent. A decrease in Cercospora leaf spot with both P. cepacia and Bt was observed. Knudsen and Spurr (1987) formulated several bacteria including B. cereus and P. cepacia into wettable powders and dusts and applied them to peanuts in the field. Limited control of Cercospora arachidicola a was achieved using P. cepacia although B. cereus survived in greater number, perhaps due to its resistant spores.
An isolate of Bacillus subtilis was found to produce a compound that inhibits urediniospore germination in Puccinia pelargonii-zonalis, the causal agent of geranium rust (Rytter and Lukezic, 1986). A culture filtrate applied to leaves prior to inoculation with the pathogen reduced the number of rust pustules by 75% compared to controls. The decrease in disease incidence resulting from the application of culture filtrate indicated antibiotic activity. A fluorescent Pseudomonas isolated from the phylloplane of cacao (Theobroma cacao) exhibited antagonism to Monilia roreri in vitro (Jimenez et al., 1986). When tested in the field it achieved control similar to that obtained with fungicide treatments. Lindow (1983) has successfully used a genetically engineered ice-minus form of Pseudomonas syringae to compete with naturally occurring ice nucleation-active bacteria and prevent frost damage. The ice-minus mutant lacks the gene necessary to produce a large surface protein that acts as an initiator for ice crystal formation. Mild frost damage, induced by nucleation-active bacteria, is a precursor for many diseases. Thus, excluding the bacteria from the microenvironment that they normally occupy reduces disease incidence.
The use of fungi as foliar biocontrol agents has received less attention than that of bacteria. There has, however, been success using Trichoderma, which produces an array of potent antibiotics, and has been shown to control Botyris bunch rot and Phomopsis viticola, the cause of dead arm disease of grape (Gullino and Garibaldi, 1986). Five applications of Trichoderma spores beginning at dormant bud stage were found to be equivalent to the control obtained by a commercial fungicide.
It is to be noted that in each of these attempts at bacteriological biocontrol of these various diseases, the introduced microorganism showed poor long-term survivability. It is likely that the limited successes achieved utilizing these techniques were probably the result of antibiotics present in the cultural filtrates coapplied to the plants with the microorganisms.
The chemical environment encountered at the surface of a plant may govern the degree of aggressiveness of virulence a pathogen exhibits. One focus of plant disease control using biological agents has been the introduction of organisms capable of preempting the nutrient supply of a pathogen (Baker and Cook, 1974). It has been shown that if an established epiphytic (foliar) ecosystem is continuously supplied with a source of nutrition, it becomes difficult for a newly introduced pathogen to compete and growth of the pathogen is limited accordingly (Warren, 1972).
Bashi and Fokkema (1977) showed that populations of Sporobolomyces roseus, an epiphytic yeast, increased when a 2% sucrose solution was applied to leaves as a food base. This indicated that the amount of nutrients present on the leaf was a limiting factor with respect to the growth of these organisms. By monitoring the growth of yeasts on wheat leaves after the application of nutrients, Fokkema et al. (1975) demonstrated that epiphytic bacteria compete effectively for nutrients. They found that yeast populations increased only after antibiotics to limit bacterial growth are applied with the nutrients. The ability of bacteria to utilize certain nutrients varies. Danilewicz (1980) found that epiphytic bacteria capable of utilizing lignin precursors were abundant where the precursors were available. The amount and type of nutrients available on the leaf surface play an important role in influencing the growth of epiphytic bacteria. By studying the effect of nutrients on the epiphytic bacteria of bean leaves, Morris and Rouse (1985) found that the application of low concentrations of nutrients affected the composition of the bacterial community by increasing the proportion of those bacteria able to utilize the nutrient applied.
In regard to disease control, addition of nutrients to aqueous bacterial suspensions enhances survival of the bacterium. Control of northern leaf blight of corn and early blight of tomato was thereby obtained in greenhouse tests (Leben and Daft, 1965). Control of Phytophthora infestans on tomato leaves in the greenhouse was achieved using water extracts from composted organic wastes (Weltzien and Ketterer, 1986). Sterile-filtered or heat-treated extracts were ineffective. The use of nitrogen-containing compounds such as urea and lecithin has also been investigated. Burchill and Cook (1971) found that in apple leaves infected with Venturia inaequalis, treatment with 5% urea resulted in the fungus producing fewer ascospores. However, this was likely a result of the urea promoting the rotting of the fallen leaves on the ground, thereby suppressing growth of the fungus. Ammonia, the breakdown product of urea, is toxic to some organisms and caused a rapid increase and significant shifts in the microbial populations from gram positive chromogens to gram negative nonchromogens. In vitro tests showed gram positive organisms stimulated growth of the scab fungus, while gram negative organisms inhibited its growth. Boudrea and Andrews (1987) had little or no success controlling V. inaequalis by the application of Chaetomium globosum ascospores to field-grown trees. The fact that few C. globosum ascospores germinate in the absence of nutrients in the form of complex media was suggested as a possible explanation for its poor performance.
With the exception of the lower fungi and yeasts, the most important structural component of the fungal cell wall is chitin (Lopez-Romero and Ruiz-Herera, 1986). In the fungal cell wall, polymers of chitin are embedded in an amorphous matrix composed of .beta.-1,3-glucans and are susceptible to degradation by chitinases in the thin primary wall formed at the growing tip of the hyphae. Ordentlich et al. (1988) in in vitro studies found that chitinolytic enzymes produced by the bacterium Serratia marcescens caused degradation of 60% of the hyphal tip cells of Sclerotium rolfsii, a soil-borne pathogen.
Chitin has been used as a soil amendment for controlling plant parasitic nematodes and soil-borne pathogens with some success. The addition of chitin to soil stimulates the growth of bacteria, actinomycetes, and fungi capable of producing chitinolytic enzymes (Brown et al., 1979; Godoy et al., 1983; Mitchell and Alexander, 1962). Godoy et al. found that the addition of chitin to soil stimulated the growth of organisms capable of degrading chitin, a component of the middle layer of egg shells in tylenchoid nematodes, through the production of chitinases. The study supported earlier results by Mian et al. (1982) that demonstrated the development of a particular soil microflora in response to the addition of chitin. Many fungi, including species of Aspergillus, Chaetomium, Fusarium, and Verticillium, showed enhanced growth after the addition of chitin to the soil and have been shown to be actively involved in cyst and egg wall degradation of species of Heterodera and Meloidogynee (Godoy et al., 1982; Godoy et al., 1983; Morgan-Jones and Rodriguez-Kabana, 1981; Ownley-Gintis et al., 1982).
In regard to the control of soil-borne fungal pathogens, Chet et al. (1986) have shown that chitin amendments stimulate the growth of the fungus Trichoderma harzianum which produces enzymes capable of degrading the cell walls of Sclerotium rolfsii, Rhizoctonia solani, and Fusarium spp., giving it great potential as a means for controlling soil-borne plant pathogenic fungi.
Each of the approaches to microbiological biocontrol of plant diseases discussed above has demonstrated only a limited success. In particular, it is noted that long term survival and reproduction of the microorganisms was not shown to be enhanced by these approaches, despite the number of years that biocontrol has been considered an ecologically and commercially important goal.